探讨粪菌移植(FMT)对严重烧伤大鼠肠道屏障功能的作用及相关机制。

按照随机数字表法将36只6～8周龄雄性SD大鼠分为3组：正常组(n＝12)、单纯烧伤组(n＝12)和FMT干预组(n＝12)。单纯烧伤组和FMT干预组大鼠制作30%总体表面积Ⅲ度烫伤模型；正常组大鼠不致伤。单纯烧伤组、FMT干预组大鼠伤后即刻腹腔注射平衡盐溶液40 mL/kg补液复苏。收集正常组大鼠新鲜粪便10 g，制成粪便滤液。伤后1 h，对FMT干预组大鼠进行粪便滤液灌胃(10 mL/kg)，间隔12 h后再次给予相同剂量粪便滤液灌胃；正常组、单纯烧伤组大鼠在相同时相点均给予等量0.9%氯化钠溶液灌胃。分别于伤后24、72 h，取伤后各组大鼠结肠内新鲜粪便各1 mL，采用实时荧光定量-聚合酶链反应检测大鼠肠道菌群属水平，即双歧杆菌、脆弱拟杆菌、乳酸杆菌、大肠杆菌、肠球菌数量；取制备好的大鼠血浆，采用荧光素异硫氰酸酯(FITC)-葡聚糖通透性实验检测3组大鼠肠道通透性；采用酶联免疫吸附试验(ELISA)检测大鼠血清中二胺氧化酶、D-乳酸及白细胞介素(IL)-6、肿瘤坏死因子(TNF)-α、IL-10水平；分别取伤后24、72 h 3组大鼠的末端回肠组织约1 cm，苏木精-伊红染色观察大鼠肠黏膜组织形态结构。数据比较采用单因素方差分析和t检验。

(1)3组大鼠在伤后24、72 h双歧杆菌、脆弱拟杆菌、乳酸杆菌、大肠杆菌、肠球菌数量比较，差异均有统计学意义(P<0.05)。伤后24 h，单纯烧伤组大鼠肠道双歧杆菌、脆弱拟杆菌、乳酸杆菌、大肠杆菌、肠球菌数量分别为(2.76±0.15)、(3.27±0.40)、(2.33±0.33)、(7.06±0.49)、(6.42±0.50) LogN/g，FMT干预组大鼠分别为(3.18±0.16)、(4.52±0.58)、(2.92±0.28)、(6.14±0.47)、(5.28±0.43) LogN/g；伤后72 h，单纯烧伤组大鼠肠道双歧杆菌、脆弱拟杆菌、乳酸杆菌、大肠杆菌、肠球菌数量分别为(3.16±0.19)、(3.79±0.42)、(2.64±0.43)、(6.34±0.56)、(5.56±0.61) LogN/g，FMT干预组大鼠分别为(3.53±0.25)、(5.50±0.32)、(3.26±0.39)、(5.37±0.70)、(4.10±0.85) LogN/g，FMT干预组双歧杆菌、脆弱拟杆菌、乳酸杆菌数量均高于单纯烧伤组，大肠杆菌、肠球菌数量均低于单纯烧伤组，差异均有统计学意义(P<0.05)。(2)伤后24 h，正常组、单纯烧伤组和FMT干预组大鼠肠道通透性分别为0.94±0.16、2.39±0.37、1.58±0.33，伤后72 h，正常组、单纯烧伤组和FMT干预组大鼠肠道通透性分别为0.94±0.17、1.88±0.57、1.21±0.24，2个时相点3组间总体比较，差异均有统计学意义(F＝34.092、7.064，P<0.05)；与单纯烧伤组比较，FMT干预组伤后24、72 h大鼠肠道通透性均降低，差异均有统计学意义(t＝ 3.971、2.664，P<0.05)。(3)伤后24、72 h，3组大鼠血清中二胺氧化酶、D-乳酸及IL-6、TNF-α、IL-10水平比较，差异均有统计学意义(P<0.05)；伤后24 h，FMT干预组大鼠血清中二胺氧化酶、D-乳酸分别为(0.93±0.13) U/mL、(3.54±0.78) μmol/L，伤后72 h分别为(0.55±1.15)U/mL、(2.58±0.51)μmol/L，均明显低于单纯烧伤组伤后24 h[(1.28±0.18)U/mL、(4.83±0.57) μmol/L]、伤后72 h[(0.86±0.21)U/mL、(4.13±0.55)μmol/L]，2组比较差异均有统计学意义(P<0.05)。伤后24、72 h，与单纯烧伤组比较，FMT干预组大鼠血清中IL-6、TNF-α水平均降低，IL-10水平均升高，差异均有统计学意义(P<0.05)。(4)伤后24、72 h，正常组大鼠肠黏膜上皮均完整，无炎症细胞浸润；伤后24 h，FMT干预组大鼠肠黏膜绒毛稍变短、排列略紊乱，散在上皮细胞破坏、黏膜脱落，有炎症细胞浸润，较单纯烧伤组相比较肠黏膜损伤减轻。伤后72 h，FMT干预组大鼠肠黏膜上皮基本完整，见少量炎症细胞浸润，肠黏膜修复较单纯烧伤组相比更为明显。

严重烧伤大鼠早期可出现肠道菌群紊乱、全身炎症反应加重、肠道屏障功能损害，FMT可以改善肠道菌群失调、抑制炎症反应，起到保护肠道屏障功能的作用。

To investigate the effect and related mechanism of faecal microbiota transplantation (FMT) on intestinal barrier function in severely burned rats.

Thirty-six male SD rats aged 6-8 weeks were divided into 3 groups according to the random number table method, normal group (n=12), pure burn group (n=12) and FMT intervention group (n=12). Thirty percent total body surface area full thickness burn model was made for rats in pure burn group and FMT intervation group, rats in normal group were not injured. Rats in pure burn group and FMT intervention group were resuscitated by intraperitoneal injection of balanced salt solution 40 mL/kg immediately after burn. Fresh faecal samples of the normal group rats were collected 10 g and made into faecal filtrate. At 1 h after injury, rats in the FMT intervention group was given faecal filtrate by gavage(10 mL/kg), and the same dose of faecal filtrate was given again 12 h later. Rats in normal group and pure burn group were given the same amount of 0.9% sodium chloride solution at the same time. At 24 and 72 h after injury, 1 mL fresh faecal samples from the colon of rats in each group were collected, and the level of intestinal flora was detected by real-time fluorescence quantification-polymerase chain reaction, namely, the number of Bifidobacteria, Bacteroides fragilis, Lactobacillus, Escherichia coli and Enterococcus. The prepared plasma of rats was taken, and luciferin isothiocyanate (FITC) -glucan permeability test was used to detect the intestinal permeability of rats in each group. The levels of diamine oxidase, D-lactic acid, interleukin (IL)-6, tumor necrosis factor (TNF)-α and IL-10 in serum were detected by enzyme-linked immunosorbent assay (ELISA). At 24 and 72 h after injury, about 1 cm of the terminal ileum tissue of rats in each group were taken respectively, and the morphological structure of intestinal mucosa was observed by hematoxylin-eosin staining. Data were processed by one-way ANOVA and t test.

(1)The differences in the number of Bifidobacteria, Bacteroides fragilis, Lactobacillus, Escherichia coli and Enterococcus in the three groups at 24 and 72 h after injury were statistically significant (P<0.05). At 24 h after injury, the numbers of Bifidobacteria, Bacteroides fragilis, Lactobacillus, Escherichia coli and Enterococcus in the pure group were (2.76±0.15), (3.27±0.40), (2.33±0.33), (7.06±0.49), (6.42±0.50) LogN/g, respectively, and those in the FMT intervention group were (3.18±0.16), (4.52±0.58), (2.92±0.28), (6.14±0.47), (5.28±0.43) LogN/g, respectively; at 72 h after injury, the numbers of Bifidobacteria, Bacteroides fragilis, Lactobacillus, Escherichia coli and Enterococcus in the pure burn group were (3.16±0.19), (3.79±0.42), (2.64±0.43), (6.34±0.56), (5.56±0.61) LogN/g, respectively, and those in the FMT intervention group were(3.53±0.25), (5.50±0.32), (3.26±0.39), (5.37±0.70), (4.10±0.85) LogN/g, the numbers of Bifidobacteria, Bacteroides fragilis and Lactobacillus in FMT intervention group were higher than those in pure burn group, and the numbers of Escherichia coli and Enterococcus in FMT intervention group were lower than those in pure burn group, the differences were statistically significant (P<0.05). (2) At 24 h after injury, the intestinal permeability of rats in normal group, pure burn group and FMT intervention group were 0.94±0.16, 2.39±0.37 and 1.58±0.33, respectively; at 72 h after injury, the intestinal permeability of rats in normal group, pure burn group and FMT intervention group were 0.94±0.17, 1.88±0.57 and 1.21±0.24, respectively, the differences among the three groups at the two phase points were statistically significant (F=34.092, 7.064; P<0.05). Compared with the pure burn group, the intestinal permeability in FMT intervention group was decreased at 24 and 72 h after injury, and the differences were statistically significant (t= 3.971, 2.664; P <0.05). (3) At 24 and 72 h after injury, the differences of the levels of diamine oxidase, D-lactic acid and IL-6, TNF-α and IL-10 in serum of the 3 groups were statistically significantly (P<0.05). At 24 h after injury, the levels of diamine oxidase and D-lactic acid in serum of FMT intervention group were (0.93±0.13) U/mL and (3.54±0.78) μmol/L, respectively, and at 72 h after injury were (0.55±1.15) U/mL and (2.58±0.51) μmol/L, respectively, which were lower than those in the pure burn group at 24 h after injury [(1.28±0.18) U/mL, (4.83±0.57) μmol/L] and at 72 h after injury [(0.86±0.21) U/mL, (4.13±0.55) μmol/L], the differences were statistically significant (P<0.05). At 24 and 72 h after injury, compared with the pure burn group, the levels of IL-6 and TNF-α in the serum of rats in the FMT intervention group decreased, and the levels of IL-10 increased, the differences were statistically significant (P<0.05). (4)At 24 and 72 h after injury, the intestinal mucosal epithelium of normal group rats was intact without inflammatory cell infiltration; at 24 h after injury, intestinal mucosal villi in the FMT intervention group was slightly shorter and disordered, scattered epithelial cells was destroyed and mucous membranes was shed, there was infiltration of inflammatory cells, compared with the pure burn group, intestinal mucosal damage was reduced. At 72 h after injury, the intestinal mucosa epithelium of the FMT intervention group was basically intact, with a small amount of inflammatory cells infiltration, and the intestinal mucosa repair was more obvious than that of the pure burn group.

The disturbance of intestinal flora, exacerbation of systemic inflammatory response and the impairment of intestinal mucosal barrier function can occur in severely burned rats at early stage, FML can improve intestinal flora disturbances, restrain inflammatory response, and protect the intestinal barrier function.